Fast axonal transport can be directly observed by light microscopy in living axons, where it is seen to consist of saltatory organelle movements. Giant axons from the lobster are particularly suitable for light microscopic study of fast axonal transport. However, the axonal plasma membrane is impermeable to many experimental probes that are used to study motility mechanisms. We have developed a technique for destroying the permeability barrier by treating it with saponin, and for reactivating organelle movement in the permeabilized axon with exogenous ATP. We propose to exploit the favorable properties of lobster giant axons, either intact, after permeabilization, or after microinjection of selected probes, in an attempt to answer major questions about the mechanisms of fast axonal transport. (1) Is fast transport based on actin and myosin, on dynein, or on some other ATPase? Monoclonal antibodies against dynein and tubulin will be used probes for dynein. Actin and myosin involvement will be tested using NEM-S1, DNAase I, brevin, and phalloidin. (2) What are the biochemical characteristics of the transport mechanism? Permeabilized axons will be used to determine the dependence of transport velocity on ATP concentration, its nucleotide specificity, and other significant parameters. Ca++/EGTA buffers will be used to analyze the role of Ca++ and other divalent cations. (3) What factors control transport? For example, we have found that EHNA, a dynein inhibitor, selectively inhibits retrograde transport. Determining the mechanism of action of EHNA may provide clues to factors that determine the direction of movement. (4) What is the ultrastructural substrate of fast transport? Intact and permeabilized axons will be studied by electron microscopy and the results correlated with the transport studies. Axons will be examined following rapid freezing and freeze-etching or freeze-substitution to obtain a view of the structure of the axoplasm that is free of the artefacts of conventional chemical fixation. (5) What are the characteristics of the axonal transport of mitochondria? (a) Measurements of velocities, direction of movement, etc., in control axons will be used to calculate the overall flow of mitochondria. (b) Mitochondria will be labeled with mitochondria-specific fluorescent dyes and fluorescence intensity, an indicator of functional state, will be correlated with mitochondrial motility. (c) Metabolic inhibitors, alterations in Ca++, and other treatments will be used to determine whether mitochondrial transport changes in response to altered physiological conditions.